By latest

Featured community

Creative Data Solutions (CDS) is a Vanderbilt Shared Resource and has extensive experience in providing effective and robust solutions to challenges pertaining to research data using modern informatics and bioinformatics approaches.

Publication

Alternative splicing of the human serotonin transporter gene.

To explore the structural basis for regulation of human serotonin transporter (hSERT) gene expression, we used primer extension and 5' rapid amplification of cDNA ends (5'RACE) techniques to estimate levels of and identify 5'-noncoding elements of hSERT mRNAs and genomic cloning to place these elements within the overall map of the hSERT gene. Primer extension on JAR cell mRNA suggested the presence of significant hSERT mRNA sequence upstream of the 5' end of our cloned hSERT cDNA. Using 5'RACE and reverse transcription-PCR (RT-PCR) methodologies, we cloned these sequences from brain and placenta and found this material to be composed of alternatively spliced exons using a previously reported noncoding exon (1A) and a novel 97-bp noncoding exon (1B). RT-PCR of JAR cell mRNA blotted with exon-specific oligonucleotides revealed both exons 1A and 1B to be regulated in a cholera toxin-dependent manner. To clarify the structure of the hSERT gene including exon 1B, we isolated and characterized genomic hSERT clones from Lambda Fix II and P1 artificial chromosome libraries. In agreement with previous findings, a single hSERT gene was identified that accounts for hybridizing bands on genomic Southern blots and was found to utilize 13 exons to encode the transporter's coding sequences along with the two noncoding 5' exons. Exon 1B was identified approximately 14 kb downstream of exon 1A in the hSERT gene and 737 bp upstream of exon 2, where the initiation site for translation is located. Exon 1B is surrounded by several elements potentially suitable for regulating serotonin transporter gene expression in vivo, including consensus sites for transcription factors AP-1, AP-2, CREB/ATF, and NF-kappaB. These data reveal additional complexity in hSERT gene structure and expression that may be relevant to regulated and compromised transporter expression in vivo.